1. Field of the Invention
The present invention relates generally to the fields of molecular biology and genetics. More particularly, it concerns the various functions of the p53 tumor suppressors and methods for identifying useful mutants having altered p53 function.
2. Description of Related Art
p53, first thought to be an oncogene, is now the most widely recognized of the class of proteins known as tumor suppressors. Its complex involvement with gene transcription, genomic stability, chromosomal segregation, senescence, cell cycle regulation and apoptosis make it one of the most important regulatory molecules now known.
Mutations in p53 have been found in cells transformed by chemical carcinogenesis, ultraviolet radiation, and several viruses. The p53 gene is a frequent target of mutational inactivation in a wide variety of human tumors and is already documented to be the most frequently mutated gene in common human cancers. It is mutated in over 50% of human NSCLC (Hollstein et al., 1991) and in a wide spectrum of other tumors.
Although the specific molecular pathway(s) through which p53 responds to DNA damage are not known, it is well known that p53 binds to DNA in a sequence specific fashion. In addition, there is considerable evidence that p53 transactivates a number of important regulatory genes including p21waf1, a potent inhibitor or most cycling-dependent kinases. Other gene products upon which p53 has some effect are GADD45, Bax, Fas, TBP, RPA, XPB, XPD and CSB.
The number of p53 molecules in a cell is limited, estimated at between 103 and 104 per cell. This relatively small number of molecules suggests some kind of post-translational regulation. One type of regulation is thought to be a reversible serine phosphorylation by at least seven distinct kinases including cdc2, casein kinase II, DNA-dependent protein kinase I, a casein kinase I-like kinase, protein kinase C, mitogen-activated kinase and JNK1. Though the precise role these different kinases play in phosphorylating p53 is not completely understood, a better understanding of the mechanisms is evolving. For example, a mutation in the casein kinase II phosphorylation site at serine 392 can reduce the antiproliferative activity of p53. Sequence specific DNA binding to p21waf1 is stimulated by phosphorylation of the serine at 315.
One of the primary goals for researchers in the p53 field has been to develop a cancer gene therapy that relies on the replacement of defective p53 genes with a wild-type p53 gene. Several clinical trials have been approved utilizing viral vectors to deliver the p53 gene and have shown notable success. Possible limitations on therapies include the amount of viral vector that is administered, duration of expression, as well as the amount of p53 being expressed in the cells. Repeated administrations also raise concerns about vector toxicity and immunogenicity.
Thus, despite the growing amount of information on p53 function, and mounting evidence of its utility in gene therapy for cancer and other hyperproliferative diseases, there remains a need for improved compositions and techniques to fully exploit p53""s remarkable biological activity.
Thus, according to the present invention, there are provided methods for identifying various modified forms of the tumor suppressor p53. In a first embodiment, there is provided a method of identifying a thermostable p53 polypeptide comprising providing a population of polynucleotides encoding activated, mutated p53 polypeptides; transforming host cells lacking an endogenous p53 polypeptide with said population of polynucleotides and culturing said host cells at elevated temperatures and under other conditions permitting the expression of said mutated, activated p53 polypeptides; screening said mutated p53 polypeptides for p53 DNA binding activity; and comparing the measured DNA binding with the DNA binding of an activated p53 polypeptide produced at said elevated temperatures, wherein increased binding of said activated, mutated p53 polypeptide, as compared to an activated p53 polypeptide, identifies a thermostable p53 polypeptide.
The method may further comprise the step of generating said population by creating random mutations in polynucleotides encoding an activated p53 polypeptide. The random mutations may be created by chemical mutagenesis, PCR mutagenesis, RT hypermutagenesis or DNA shuffling. In a particular embodiment, the activated p53 polypeptide contains a truncation of a C-terminal portion of wild-type p53, for example the 30 C-terminal amino acid residues. Alternatively, the truncation may specifically encompass residue 360, or one or more point mutations when compared to wild-type p53. In another embodiment, the activated p53 molecule may contain an insertion as compared to wild-type p53, or a substitution in the C-terminus of wild-type p53. In yet another embodiment, the activated p53 polypeptide comprises an internal deletion.
The host cells may be bacterial cells, for example, Escherichia coli. Alternatively, the host cells may be eukaryotic cells, for example, yeast cells. The binding activity may be determined using a labeled target DNA, for example, where label is a radiolabel, a chemilluminescent label or a fluorescent label. The elevated temperature may be 37xc2x0 C.
Also provided are thermostable p53 polypeptides comprising a first point mutation. Throughout the application, reference to a particular residues of p53 is made by reference to SEQ ID NO:2. The first point mutation may be Val133, Tyr239, Asp268, Val336, Pro364, Val62, Thr116 Pro166, Th270, Ser88, Ile157, Val344, Gly42, Ser268, Lys51, Gly326, Glu207, Ser212, His264, Ala203, Leu80, Ala30, Lys56, Asn106, Arg115, Ser277, Met344, Gln45, Ala102, Ser191, Thr322, Ala31, Gly49, Thr183, Ile264 or Val346. The polypeptide may further comprise a second point mutation. The combinations may be where said first point mutation is Gly42 and said second point mutation is Ser268, where said first point mutation is Lys51 and said second point mutation is Gly326, where said first point mutation is Leu80 and said second point mutation is Ala203, where said first point mutation is Ser277 and said second point mutation is Met344.
The polypeptide may further comprise a third point mutation. The combinations may be where said first point mutation is Tyr239, said second point mutation is Asp269, and said third point mutation is Val336; where said first point mutation is Val62, said second point mutation is Tyr239, and said third point mutation is Asp268; where said first point mutation is Asp268, said second point mutation is Val336, and said third point mutation is Pro364; where said first point mutation is Ser88, said second point mutation is Ile157, and said third point mutation is Val344; or where said first point mutation is Glu207, said second point mutation is Ser212, and said third point mutation is His364. The polypeptide may further comprise a fourth point mutation. The combinations may be where said first point mutation is Thr116, said second point mutation is Pro166, said third point mutation is Asp268, and said fourth point mutation is Thr270.
The polypeptide may further comprise a fifth point mutation. The combinations may be where said first point mutation is Val133, said second point mutation is Tyr239, said third point mutation is Asp268, said fourth point mutation is Val336, and said fifth point mutation is Pro364; where said first point mutation is Ala30, said second point mutation is Lys56, said third point mutation is Asn106, said fourth point mutation is Arg115, and said fifth point mutation is Ala203; where said first point mutation is Gln45, said second point mutation is Ala102, said third point mutation is Ser191, said fourth point mutation is Glu207, and said fifth point mutation is Thr332; or where said first point mutation is Ala31, said second point mutation is Gly49, said third point mutation is Thr183, said fourth point mutation is Ile264, and said fifth point mutation is Val346.
In another embodiment, there is provided a method of identifying an activated p53 polypeptide comprising providing a population of polynucleotides encoding mutated p53 polypeptides; transforming bacterial host cells lacking an endogenous p53 polypeptide with said population of polynucleotides and culturing said host cells under conditions permitting the expression of said mutated p53 polypeptides; screening said mutated p53 polypeptides for p53 DNA binding activity; and comparing the measured DNA binding of step with the DNA binding of wild-type p53, wherein increased binding of a mutated p53 polypeptide, as compared to wild-type p53, identifies an activated p53 polypeptide.
The method may further comprise the step of generating said population by creating random mutations in polynucleotides encoding wild-type p53, for example, by chemical mutagenesis, PCR mutagenesis, RT hypermutagenesis or DNA shuffling.
Again, the bacterial host cells may be Escherichia coli. The screening for DNA binding activity may comprise lysing said host cells and contacting the lysates with a target DNA to which activated p53 binds. The target DNA may be labeled.
In yet another embodiment, there is provided an activated p53 polypeptide. The polypeptide may comprise a deletion of residue 360, but retain sequences flanking residues 360. Alternatively, the polypeptide may comprise a first point mutation when compared to wild-type p53, such as Lys3, Arg23, Ser54, Asn106, Ala123, Arg137, Thr159, Thr160, Asp268, Ser268, Thr332, Gly339 or Val344. The polypeptide may comprise a second point mutation. The combinations may be where said first point mutation is Leu77 and said second point mutation is Ala122. The polypeptide may comprise a third point mutation. The combinations may be wherein said first point mutation is Leu4, said second point mutation is Ala225, and said third point mutation is Ser310. Alternatively, the polypeptide may comprise an insertion when compared to wild-type p53, for example, an insertion at residue 360. The polypeptide may also comprise, in addition to an insertion, an internal deletion, such as, where said insertion is at residue 317 and said deletion is at residue 365.
In still yet another embodiment, there is provided a method of identifying an activator of p53 DNA binding comprising providing a plurality of cDNAs and a polynucleotide encoding a full length p53 polypeptide; transforming host cells lacking an endogenous p53 polypeptide with said cDNAs and said p53-encoding polynucleotide, and culturing said host cells under conditions permitting the expression of products encoded by said cDNAs and said full length p53 polypeptide; and screening said products encoded by said cDNAs for p53 DNA binding activity.
In yet a further embodiment, there is provided a method of identifying an inhibitor of p53 DNA binding comprising providing a plurality of cDNAs and a polynucleotide encoding an activated p53 polypeptide; transforming host cells lacking an endogenous p53 polypeptide with said cDNAs and said p53-encoding polynucleotide, and culturing said host cells under conditions permitting the expression of products encoded by said cDNAs and said activated p53 polypeptide; and screening said products encoded by said cDNAs for loss of p53 DNA binding activity.
In still yet a different embodiment, there is provided a method of identifying SV40 resistant p53 polypeptides comprising providing a polynucleotide encoding the SV40 large T antigen and a population of polynucleotides encoding mutated, activated p53 polypeptides; transforming host cells lacking an endogenous p53 polypeptide with said SV40 large T antigen-encoding polynucleotide and said p53-encoding polynucleotides, and culturing said host cells under conditions permitting the expression of said SV40 large T antigen and said mutated, activated p53 polypeptides; screening said mutated, activated p53 polypeptides for p53 DNA binding activity; and comparing the measured DNA binding with the DNA of an activated p53 polypeptide coexpressed with SV40 large T antigen, wherein increased binding of a mutated, activated p53 polypeptide, as compared to activated p53, identifies an SV40 resistant p53 polypeptide.
In another embodiment, there is provided a method for identifying a p53 polypeptide with increased transcriptional activity comprising providing a population of mutated p53 polypeptides; transforming host cells lacking an endogenous p53 polypeptide with said population of mutated p53 molecules, wherein said host cells contain a reporter gene driven by a p53-dependent promoter; screening said host cells for expression of the gene product encoded by said reporter gene; and comparing the measured expression of said reporter gene with the expression of said reporter gene by wild-type p53, wherein an increase in the expression of said reporter gene in cells expressing said mutated p53 molecules, as compared to cells expressing wild-type p53, indicates a p53 polypeptide having increased transcriptional activity.
The reporter gene may encode a fluorescent polypeptide and said screening comprises fluorescence activated cell sorting. The host cells may be osteocarcinoma cells and said transforming may comprise retroviral infection with a population of retroviruses encoding said mutated p53 polypeptides. The method may further comprise the step of PCR amplification of an identified p53 polypeptide having increased transcriptional activity.
In yet a different embodiment, there is provided a mutant p53 polypeptide containing an HIV-1 protease cleavage site upstream of the carboxy-terminal region thereof, whereupon cleavage by HIV-1 protease, said p53 molecule is activated. The polypeptide may comprise all of the wild-type p53 residues in addition to said protease cleavage site, or the cleavage site may replace a similar sized region of the wild-type p53 polypeptide. The cleavage site, in a particular embodiment, is VSFNFPQITL. The cleavage site may be inserted immediately after amino acid residue 359. The amino acid sequence VSFNFPQITL may be substituted for amino acid residues 360-369 of the wild-type p53 amino acid sequence.
Another aspect of the invention is an infectious retrovirus, the RNA of which encodes a mutant p53 polypeptide containing an HIV-1 protease cleavage site upstream of the carboxy-terminal region thereof, whereupon cleavage by HIV-1 protease, said p53 molecule is activated. Preferably, the mutant p53 coding region is under the control of the viral LTR.
Still another aspect of the invention is a method of inhibiting HIV-1 replication in a cell infected with HIV-1 comprising contacting said cell with an infectious retrovirus, the RNA of which encodes a mutant p53 polypeptide containing an HIV-1 Tat protease cleavage site upstream of the carboxy-terminal region thereof, whereupon cleavage by HIV-1 Tat, said p53 molecule is activated. The method may further comprise treatment of said cell with AZT or with HAART.
Still yet another aspect of the invention is a polynucleotide encoding a thermostable p53 polypeptide comprising a first point mutation selected from the group consisting of Val133, Tyr239, Asp268, Val336, Pro364, Val62, Thr116, Pro166, Thr270, Ser88, Ile157, Val344, Gly42, Ser268, Lys51, Gly326 Glu207, Ser212, His264, Ala203, Leu80, Ala30, Lys53, Asn106, Arg115, Ser277, Met344, Gln45, Ala102, Ser191, Thr322, Ala31, Gly49, Thr183, Ile264, and Val346. Another polynucleotide according to the invention encodes an activated p53 polypeptide comprising of a deletion of residue 360, but retaining sequences flanking residues 360. Still another polynucleotide according to the invention encodes an activated p53 polypeptide comprising a first point mutation when compared to wild-type p53. Still yet another polynucleotide of the invention encodes an activated p53 polypeptide comprising an insertion when compared to wild-type p53, and further may comprise a deletion.